Based on a complementary approach employing recombinant DNA strategies, and modern genetic analyses, the proposed research program aims to identify and describe the molecular mechanisms whereby cells respond to environmental challenges via gene amplification. Our studies center on copper toxicity in yeast. Genetically tractable commercial and laboratory yeast strains characterized by single copy and stably iterated CUP1 genes will be assessed for their capacity to achieve gene amplification under defined selective conditions in wholly defined media. DNA sequence comparisons between strains known to carry distinctively different iteration units should generate critical information impinging on our proposed misaligned pairing and crossing over model, a scheme that readily generates tandem duplications from progenitor single copy genes. Generating two gene copies from one may comprise the rate limiting step, since amplification starting from two or more units to a larger number occurs frequently, i.e. in 50% of resistant outgrowths on selective media. Our preliminary studies suggest an upper limit to the number of stable tandem repeats that can be organized and maintained within the CUP1 locus of chromosome VIII. Since phenotypic resistance levels may increase under selection, without concomittant amplification at the normal resident site, we shall determine whether additional gene copies are displaced to other genomic sites. If mobile sequences are identified, analysis of the control and regulation of mobility will be justified. By integrating our plasmids containing known numbers of repeat units into predetermined chromosomal regions, we are empowered to study the effect of specific regions marked by high or low mitotic recombination, high or low interference on gene amplification, and possibly on sister chromatid exchange and intrachromosomal gene conversion. Also, we shall investigate the effect of genetic substitutions such as rem1, spo11 and rad52 on the amplification process. Finally we shall delineate the rules of gene conversion when sequences with known but different iterations undergo recombination in mitotic and meiotic cells. Molecular analyses at the DNA and RNA levels will be coordinated with appropriate genetic studies throughout.